The Matrix Biochemistry Unit has been directed by Dr Larry W. Fisher directly working with Dr. Abdullah Karadag (Research Fellow, VP), Dr. Kalu Ogbereke (Research Fellow), Ms Li Li (Technical IRTA) and Dr. Neal S. Fedarko (an IPA with Johns Hopkins University). The Unit has continued to focus on structure-function studies of the noncollagenous proteins of bones and teeth, with particular emphasis on matrix protein-protein and matrix protein-cell interactions. It is highly likely that both the assembly of the matrix and its subsequent mineralization are controlled by cells via the use of noncollagenous proteins. Other noncollagenous proteins are likely used in cell-cell and cell-matrix signal transduction. We have started projects studying the roles that some of the ?bone and tooth? matrix proteins may play in normal ducts of the salivary gland and kidney as well as continued our projects on their roles in the development and metastasis of tumors. This year we have reached the intermediate goals in several areas discussed below. We have continued to present evidence to support our hypothesis that five small integrin-binding proteins [bone sialoprotein (BSP), osteopontin (OPN), dentin matrix protein 1 (DMP1), dentin sialophosphoprotein (DSPP) and matrix extracellular phosphoglycoprotein (MEPE)] whose genes are clustered within 375,000 base pairs on human chromosome 4 (mouse chromosome 5) are a gene family with related biological functions. We have named this family the SIBLINGs (Small Integrin-Binding LIgand, N-linked Glycoproteins.) All of these proteins are clearly expressed in bones and teeth and were thought to be rarely expressed outside of the skeleton. The Unit is currently challenging this concept by documenting the presence of all five gene products in several normal epithelial tissues including kidney and salivary gland. For example, the SIBLINGs and their protein-cutting partners, the matrix metalloproteinases (MMP), are all expressed in the ductal structures of human salivary glands. We have theorized that these proteins play a critical in the long-term maintenance of these ducts that modify the composition of the saliva before it enters the oral cavity. These proteins will also likely serve as useful identifiers of mature saliva ductal cells in our colleagues? future salivary gland stem cell cloning and other cell culture projects. Another, major breakthrough this year was our evidence that three members of the SIBLING family (BSP, OPN and DMP1) bind and activate three different members of the matrix metalloproteinase (MMP) family of protein-digesting enzymes (MMP-2, MMP-3 and MMP-9 respectively). The ability of the SIBLINGs to turn on the protease activity without removing the inhibitory propeptide is a new paradigm in the activation of MMPs. The same SIBLING/MMP pairs are also resistant to inhibition by both natural (tissue inhibitors of matrix metalloproteinases, TIMPs) and synthetic inhibitors of MMP activity. These observations add a new dimension to the current working models of how cells make, modify, and remove protein complexes found outside of cells. These data also offer a possible explanation of why recent cancer drug trials of compounds thought to inhibit MMP activity in cancer patients may have been unsuccessful. In the presence of the appropriate SIBLING proteins, the proteases targeted by these drugs may not be inhibited as planned and remain active. Furthermore, we have shown that many cancer cells in tissue culture can use BSP and MMP-2 on their cell surfaces to more effectively move though an artificial barrier thought to mimic barriers encountered by cancer cells as they attempt to move throughout the body. This may help explain, in part, why breast cancer patients with high levels of BSP in their tumors tend to have a shorter life expectancy than those whose tumors do not express this SIBLING. The Unit has successfully ccontinued our studies into the use of SIBLINGs as serum markers of various metabolic conditions and diseases. The SIBLING, MEPE, was originally discovered by others to be associated with oncogenic hypophosphotemic osteomalacia in which a small tumor releases hormones that then cause harmful losses of phosphate ions in the urine. This year, using an assay that was developed with our colleagues at Johns Hopkins University, we have shown serum MEPE levels were significantly correlated with serum phosphorus and parathyroid hormone (PTH) levels as well as with hip bone mineral density in normal volunteers. These results are consistent with MEPE being involved in phosphate and bone mineral metabolism. Whether or not this now well-defined serum assay is useful in the detection or progression of various bone and kidney disorders remains to be tested in future years. We also developed an assay to accurately quantify the levels of the somatic gene mutations in individual lesions of fibrous dysplasia/McCune-Albright patients that are studied by our colleagues in our Branch. With this assay, colleagues may be able to decipher some of the interactions of normal and mutant cells in the formation, progression and decline of the FD/MAS lesions. The same technique can probably be used to quantify any known mutation within samples of fresh, frozen or paraffin-embedded tissues. This technique may have particularly interesting uses in quantifying a number of previously described mutations in tissues as they progress from normal tissue, through dysplastic lesions, to malignant tumors. The Matrix Biochemistry Unit freely gives probes (antisera, cDNA, proteins etc.) to any laboratory in the world that makes a reasonable request. In FY04 we sent ~300 probes to 125 laboratories (14% were in the dental field) around the world. Others in the Branch have sent these same probes during this time as well.